1. Technical Field
The present invention relates to assays and panels for detection of biomarkers, with the term “biomarker” referring to an analyte in a body fluid that is associated with a physiological condition and/or the presence or risk of contracting one or more diseases. In particular, the present invention relates to non-invasive detection of biomarkers in urine.
2. Background Art
It is well established in the scientific literature that certain physiological conditions, including oxidative stress and/or chronic inflammation, play key roles in several pathological disturbances such as atherosclerosis, obesity, diabetes, neurodegenerative diseases and cancer. Diet, lifestyle, exercise, as well as certain drugs have anti-inflammatory and/or anti-oxidant activity. Indeed, the market for antioxidants alone runs to billions of dollars per year. Many biomarkers for inflammation, oxidative stress, and anti-oxidant activity have been reported in the literature.
In contrast to the assessment of wellness or relative health, or for the assessment of the risk of development of disease(s), traditional tests are designed and employed to diagnose specific diseases, with an increasing emphasis on early diagnosis. Some available tests do analyze for some substances, such as cholesterol, lipoproteins, and CRP (c-reactive protein), albumin/creatinine ratio, and some other “risk factors” for specific diseases, e.g. cardiovascular disease. But, the disease-specific application of these few pre-symptomatic tests is still consistent with traditional medicine's focus on biomarkers for the diagnosis of specific disease
For example, although chronic inflammation is associated with a significant increase in the risk for certain cancers, and regular use of drugs or dietary agents with anti-inflammatory activity have been proven to reduce the risk for such cancers, traditional clinical laboratories and clinicians do not monitor biomarkers for inflammation as risk factors for cancer.
Some “esoteric laboratories” offer a large number of tests such as cytokine assays, mostly using blood samples, to test for many reported biomarkers associated with disease(s) or disease risk. A few internet-based companies offer products that are purported to provide for the qualitative determination of oxidative stress biomarkers such as TBARS (thiobarbituric acid reactive substances) or other tests for biomarkers associated with oxidative stress (e.g. isoprostanes) in urine.
However, with the exception of the disease-specific (almost exclusively related to cardiovascular disease) application of the few examples cited above, at present none are readily available to individuals seeking to determine how healthy (low inflammation, low oxidative stress, high antioxidant activity) they are. As a specific example, the currently available CRP test only interprets the level of CRP as a marker for cardiovascular risk.
A few companies offer a wide range of exotic tests for human physiological biomarkers. For example, Genova Diagnostics offers an inflammation panel comprised of 3 inflammatory biomarkers (hsCRP, homocysteine and fibrinogen) in a blood sample, and an Oxidative Stress 2.0 blood test panel comprised of 10 biomarkers, one of which is lipid hydroperoxides. However, typically these tests are run either individually or in panels on blood samples and almost always require the samples be sent to a core laboratory. The latter requirement introduces several undesirable characteristics, including: the time, effort and cost of collection and transport of the specimens, the significant potential for ex vivo changes in the level(s) of the analytes that may arise either from the decomposition of an analyte or the artifactual generation of additional analyte from precursors in a sample. Such artifactual ex vivo changes in the levels of analytes are particularly well known in the case of oxidative stress biomarkers, but can also occur for inflammatory biomarkers in blood or urine specimens. For example, isoprostanes, which are well-studied biomarkers of oxidative stress, are rapidly generated ex vivo by the action of reactive oxygen species on arachidonic acid present in blood samples; and the level of protein in a urine sample may artifactually increase within hours at room temperature due to bacterial growth.
For example, U.S. Pat. No. 6,953,666 to Kinkade, Jr., et al. discloses methods and compositions for detecting the presence of oxidized derivatives of amino acids in proteins as biomarkers of oxidative stress. In principle, the biomarker can be any amino acid that has undergone oxidation (or other modification, e.g. dityrosine, nitrotyrosine which is produced by the reaction of tyrosine with peroxynitrite, or chloro-tyrosine, which is produced by the action of myeloperoxidase and is an inflammatory biomarker). Emphasis in Kinkade, Jr., et al. is given to oxidized sulfur- or selenium-containing amino acids (SSAA). Oxidized SSAA are amino acids in which the sulfur or selenium moiety has been oxidized to some oxidation state. Oxidized SSAA include, but are not limited to, cysteine, cystine, methionine, selenomethionine, selenocystine and selenocysteine in their various possible oxidation states. Typically, an ELISA assay is provided for quantification of these biomarkers.
U.S. Pat. No. 6,852,541 to Obayan, et al. discloses an assay for testing oxidative stress of a subject by measurement of oxidants in biological fluids such as urine, plasma, bioreactor medium and respiratory aspirants. There is provided a method of determining oxidative stress in a mammalian subject. The method comprises: obtaining a sample of a biological fluid from the subject; mixing the biological fluid with a ferrous reaction reagent; incubating the biological fluid and the reaction reagent; and detecting a colored reaction product. There is further provided a ferrous reaction reagent suitable for use in assaying oxidative stress, said reaction reagent comprising 2-deoxyglucose, TBA, EDTA, and ferrous sulfate, and being substantially free of ascorbic acid.
U.S. Pat. No. 7,288,374 to Pincemail, et al. discloses a process for detecting oxidative stress in a sample and to a kit for this implementation. According to one embodiment, the Pincemail, et al. invention provides a method for the detection of oxidative stress in an individual carrying a risk factor for oxidative stress comprising determining the risk factor for oxidative stress of said individual; selecting at least two oxidative stress markers being increased or decreased for said risk factor relative to healthy individuals; and measuring the amount of said at least two oxidative stress markers in a sample obtained from said individual. Oxidative stress markers in the invention of Pincemail, et al. are detected from whole blood samples or samples containing components thereof.
U.S. Pat. No. 5,858,696 to Roberts, II et al. discloses a method of assessing oxidative stress in vivo by quantification of prostaglandin F2-like compounds and their metabolites produced by a non-cyclooxygenase free radical catalyzed mechanism.
U.S. Pat. No. 5,912,179 to Alvarez, et al. discloses systems and methods for material analysis in which an organic sample (e.g., a foodstuff, tissue sample or petroleum product) is illuminated at a plurality of discrete wavelengths that are absorbed by fatty acid and fatty acid oxidation products in the sample. Measurements of the intensity of reflected or absorbed light at such wavelengths are taken, and an analysis of absorbance ratios for various wavelengths is performed. Changes in the reflection ratios are correlated with the oxidative state of fatty acids present in the material.
U.S. Pat. Nos. 6,096,556 and 6,133,039 disclose a non-invasive method for the determination of oxidative stress in a patient by urinalysis. The method comprises quantifying the level of o,o′-dityrosine in a sample of the urine of the patient and comparing with the corresponding level of the compound in a normal or control sample, whereby a substantially elevated level of said o,o′-dityrosine is indicative of oxidative stress in the patient.
U.S. Pat. No. 6,541,265 to Leeuwenburgh discloses methods and systems for testing a substance for inflammatory or oxidant properties under acute inflammatory conditions characterized by increased levels of redox-active metal ions. The method includes the steps of applying an eccentric exercise stimulus to a subject, thereby inducing a muscle injury; administering a substance of interest to the subject; measuring one or more biological markers of inflammation, oxidative stress, and muscle damage, or combinations thereof, within the subject; and correlating the measured value of the biological marker(s) with the inflammatory or oxidative properties of the substance administered. The systems of the subject invention include means for obtaining a biological sample from a subject, means for applying eccentric exercise stimulus to the subject; means for measuring the amount of the biological marker(s) within the biological sample; and means for correlating the measured amounts of the biological marker(s) with the inflammatory or oxidant properties of the substance administered.
U.S. Pat. No. 6,569,683 to Ochi, et al. discloses a diagnostic plot derived from the measurement of 82 assays that characterize two key parameters that significantly contribute to an individual's health status. These two parameters are oxidative stress profile (OSP) and antioxidant profile. Each of the 82 assays is complimentary with other assays of the profile, thus providing either confirmation information or the synthesis of new information. The diagnostic plot, developed to interpret the assay data, which provides information about oxidative damage and antioxidant protection, consists of four quadrants, each with noticeable characteristics. By visually assessing the position of a patient's OSP status, in comparison to reference OSP values in the four quadrants constituting the diagnostic plot, physicians and other health care professionals can provide sound advice to their patients regarding dietary and life style changes one need to adhere for prevention of oxidative stress-related diseases as well as postponing premature aging processes.
Vassalle et al. (Vassalle C, Pratali L, Boni C, Mercuri A, Ndreu R. An oxidative stress score as a combined measure of the pro-oxidant and anti-oxidant counterparts in patients with coronary artery disease. Olin Biochem. 41:1162-7 (2008)) have report an “oxidative stress index” in which tests for both the oxidative damage and antioxidant components of a blood sample are performed and the Oxidative-INDEX is computed based on a formula employing both components.
U.S. Patent Application Publication No. 2007/0054347 to Rosendahl, et al. discloses an optical analyzer for measuring an oxidative stress component in a patient, having a light source and a light detector used for measuring an optical property of a medium and generating optical measurement data. A processor analyzes the optical measurement data and generates a value for one or more oxidative stress component in the form of a redox signature for the patient. Probability data of the presence of an oxidative stress dependent disease can be calculated. By observing at least one additional clinical condition of the disease, a diagnosis using said at least one additional condition and said redox signature can be obtained.
U.S. Patent Application Publication No. 2010/0267037 to Westbrook, et al. discloses a method for detection of inflammatory disease in a subject that comprises assaying a test sample of peripheral blood from the subject for a marker of DNA damage. An elevated amount of the marker present in the test sample compared to control sample and this is described to be indicative of inflammatory disease activity, including sub-clinical inflammation. The method can be adapted for quantitatively monitoring the efficacy of treatment of inflammatory disease in a subject. Markers of DNA damage include single- and/or double-stranded breaks in leukocytes, oxidative DNA damage in leukocytes, or a marker of nitric oxide oxidative activity (protein nitrosylation in leukocytes). The inflammatory disease can be inflammatory bowel disease (ulcerative colitis or Crohn's disease). The invention is described as also being useful for detection of other types of inflammatory disease, such as non-immune intestinal inflammatory disease (diverticulitis, pseudomembranous colitis), autoimmune diseases (rheumatoid arthritis, lupus, multiple sclerosis, psoriasis, uveitis, vasculitis), or non-immune lung diseases (asthma, chronic obstructive lung disease, and interstitial pneumonitis).
The methods cited above typically require complex instrumentation and technically skilled operator, so that they are expensive and not suitable for widespread application. Further, as noted above, this typically requires that samples be transported to specialized locations capable of performing such analyses, which may result in alterations to the analyte(s).
Many devices have been developed to analyze for specific substances in biological specimens at the point of testing by employing dry chemical, microfluidic and/or immunochemical methods. Several such methods, which are in widespread use, are essentially dry chemistry tests involving test pads into which chemicals have been impregnated and which react relatively specifically with analytes in with biofluids, and the results of which can be read by optical or other methods. The analysis can involve simply visual comparison to the color of a reference chart, which is widely employed for the qualitative analysis of water in pools and spas and for the analysis of multiple disease-related analytes in urine and other body fluids. Semi-quantitative results may be obtained by the application of a device to measure the amount of color developed.
For example, U.S. Pat. No. 5,597,532 to Connolly discloses an apparatus for the optoelectronic evaluation of test paper strips for use in the detection of certain analytes in blood or other body fluids. The test strip comprises an elongated plastic part including a hinged portion to allow a first portion to be folded over a second portion. A series of layers of test strips are disposed between the folded over portions of the test strip. The test strip is configured such that the chemistry layers are placed in contacting engagement with one another, but not compressing one another. A reflectance photometer is provided and includes various features, including a lot number reader wherein if the test strip does not match the memory module, a test is not performed, and the user is instructed to insert a correct memory module.
U.S. Pat. Nos. 6,511,814 and 6,551,842 to Carpenter discloses a disposable, dry chemistry analytical system that is broadly useful for the detection of a variety of analytes present in biological fluids such as whole blood, serum, plasma, urine and cerebral spinal fluid. The invention discloses the use of the reaction interface that forms between two liquids converging from opposite directions within a bibulous material. The discovery comprises a significant improvement over prior art disposable, analytical reagent systems in that the detectable reactant zone is visually distinct and separate from the unreacted reagents allowing for the use of reaction indicators exhibiting only minor changes as well as extremely high concentrations of reactants. In addition, staged, multiple reagents can be incorporated. Whole blood can be used as a sample without the need for separate cell separating materials. Finally, the invention is useful for the detection of analytes in a broad variety of materials such as milk, environmental samples, and other samples containing target analytes.
U.S. Pat. No. 7,267,799 to Borich, et al. discloses an optical reading system, a universal testing cartridge, and a method of coupling optical reading systems. In a particular illustrative embodiment, the optical reading system includes a universal test cartridge receptor, test format determination logic, test criteria determination logic, and an optical reader module. The universal test cartridge receptor is responsive to a universal test cartridge having a test strip inserted therein. The test format determination logic determines an optical test format of the test strip. The test criteria determination logic determines an optical test criteria based upon the optical test format. The optical reader module is configured to capture an optical test image of the test strip.
U.S. Pat. No. 7,425,302 to Piasio, et al. discloses a lateral flow chromatographic assay format for the performance of rapid enzyme-driven assays. A combination of components necessary to elicit a specific enzyme reaction, which are either absent from the intended sample or insufficiently present therein to permit completion of the desired reaction, are predeposited as substrate in dry form together with ingredients necessary to produce a desired color upon occurrence of the desired reaction. The strip is equipped with a sample pad placed ahead of the substrate deposit in the flowstream, to which liquid sample is applied. The sample flows from the sample pad into the substrate zone where it immediately reconstitutes the dried ingredients while also intimately mixing with them and reacting with them at the fluid front. The fluid front moves rapidly into the final “read zone” wherein the color developed is read against predetermined color standards for the desired reaction. Pretreatment pads for the sample, as needed, (e.g. a lysing pad for lysing red blood cells in whole blood) are placed in front of the sample pad in the flow path as appropriate. The assay in the format of the invention is faster and easier to perform than analogous wet chemistry assays. Specific assays for glucose-6-phosphate dehydrogenase (“G-6PD”), total serum cholesterol, .beta.-lactamase activity and peroxidase activity are disclosed.
U.S. Pat. No. 7,521,260 to Petruno, et al. discloses an assay test strip includes a flow path, a sample receiving zone, a label, a detection zone that includes a region of interest, and at least one position marker. The at least one position marker is aligned with respect to the region of interest such that location of the at least one position marker indicates a position of the region of interest. A diagnostic test system includes a reader that obtains light intensity measurement from exposed regions of the test strip, and a data analyzer that performs at least one of (a) identifying ones of the light intensity measurements obtained from the test region based on at least one measurement obtained from the at least one reference feature, and (b) generating a control signal modifying at least one operational parameter of the reader based on at least one measurement obtained from the at least one reference feature.
U.S. Patent Application Publication No. 2009/0155921 to Lu, et al. discloses a method and apparatus for reading test strips such as lateral flow test strips as used for the testing of various chemicals in humans and animals. A compact and portable device is provided that may be battery powered when used remotely from the laboratory and, may store test data until it can be downloaded to another database. Motive power during scanning of the test strip is by means of a spring and damper that is wound by the operator during the insertion of a test strip cassette holder prior to test.
U.S. Patent Application Publication No. 2010/0311181 to Abraham, et al. discloses an assay reader system incorporating a conventional assay reader, for example a lateral flow reader, and an insert aligned with the readers sensor to detect an assay result. The insert may include a housing that defines a cavity to receive a removable barrier, wherein the removable barrier can be aligned between the sensor and the test strip. The barrier may include an optical window, and may be cleanable and/or disposable to maintain the accuracy of the reader. Test strips are introduced into the reader through a receiving port within the insert's housing. An air inlet on the insert further maintains the readers accuracy by allowing air to be tunneled over the housing to remove excess dust, debris, or the like.
The current methods described above for the assessment of oxidative stress, antioxidant capacity and inflammation have multiple drawbacks, including: some of the biomarkers (such as most oxidized lipids) are not stable for prolonged periods, even when stored frozen; some biomarkers (e.g. isoprostanes, widely regarded as biomarkers for oxidative stress) are generated ex vivo from the precursor (arachidonic acid) when some biological samples (particularly blood) are exposed to oxygen in the air; most require blood, which is invasive and requires a skilled person to collect the sample; most of the exotic testing laboratories have very high fees so that a multi-analyte assessment of healthy may cost from $2,000 to over $10,000, and typically requires a physician to analyze and interpret the data. Furthermore, some available tests, such as a commercial test marketed for monitoring lipid hydroperoxides in urine (it should be noted: free radicals themselves are so short-lived that they can't be directly measured in biofluids), do not employ any method to adjust or normalize the analysis for the relative concentration of the urine sample.
Furthermore, the levels of many of the biomarkers employed to assess oxidative stress, inflammation and/or antioxidant activity are impacted by and respond rapidly to factors unrelated to an individual's overall health and risk for contracting diseases. For example, the level of reactive oxygen species and consequently the levels of many biomarkers for oxidative stress, including isoprostanes and malondialdehyde, increase rapidly albeit transiently as a consequence of physical exercise. The level of nitric oxide metabolites (nitrate and nitrite) are transiently elevated following the consumption of processed foods containing nitrates as preservatives. The levels of urinary proteins can also be elevated by physical exercise. The level of isoprostanes in the urine is further influenced by the rapid metabolism of isoprostanes by the body, with the mechanism(s) and extent of metabolism of isoprostanes subject to considerable variation among individuals. Since uric acid is one of the major antioxidants present in blood and urine, the antioxidant activity of a sample is subject to variations in the rate of purine catabolism and also to dietary factors. For example, it has been reported that the primary mechanism responsible for the increase in antioxidant activity following consumption of apples is the uric acid derived from the apples. Hence, although there is significant evidence that the levels of specific individual biomarkers for oxidative stress, inflammation and/or antioxidant activity are related to health and disease risk based on extensive studies in experimental animals and in human populations, confounding factors such as those listed above are among the reasons why the application of these biomarkers for the assessment of the health and disease risk of individual humans has been very restricted.
Therefore, there is a need for a set of tests to quantify these biomarkers for these important physiological conditions, preferably including multiple biomarkers to significantly reduce confounding effects associated with the use of a single biomarker, that signal an individual's health and relative resistance to multiple diseases that can preferably be performed non-invasively for low cost and can provide accurate results regarding the health of the user.